Duchenne muscular dystrophy (DMD) is the most frequent progressive muscle degenerative disease, affecting approximately one in 3,500 to 5000 male births. DMD is caused by deletions or mutations in the gene encoding dystrophin, located on the X chromosome. Dystrophin is required for the assembly of the dystrophin-glycoprotein complex, and provides a mechanical and functional link between the cytoskeleton of the muscle fiber and the extracellular matrix. The absence of functional dystrophin causes fiber degeneration, inflammation, necrosis and replacement of muscle with scar and fat tissue, resulting in progressive muscle weakness and premature death due to respiratory and cardiac failure between the second and fourth decade of life (Moser, H., Hum Genet, 1984. 66(1): p. 17-40).
A milder form of the disease called Becker muscular dystrophy (BMD) is distinguished from DMD by delayed onset, later dependence on wheelchair support, and longer life span. BMD is caused by mutations maintaining the reading frame and the most critical parts of the gene, leading to a truncated but still functional dystrophin protein (Muntoni F et al, Lancet Neurol, 2003).
There is no cure nor effective treatment available for DMD (Rodino-Klapac, L. R. et al., Curr Neurol Neurosci Rep, 2013. 13(3): p. 332) or BMD. Conventional therapies are limited to supportive care, which partially alleviates signs and symptoms, but does not directly target the disease mechanism nor reverse the phenotype.
There currently are several therapeutic strategies being developed for DMD including in vivo gene therapy, cell transplantation therapy, pharmacologic rescue of DMD nonsense mutations and exon skipping strategies to repair the DMD gene reading frame. All of these strategies have problems to overcome, including targeting different muscle groups, optimization of delivery, long-term expression of the transgene, and potential immune response (Jamin et al., Expert Opin Biol Ther, 2014).
The dystrophin gene is the largest known gene in the human genome and is too large to fit inside known gene therapy vector systems. Therefore, as of today, there are essentially two gene therapy strategies for DMD with viral vectors: i) constitutive expression of antisense oligonucleotides to promote exon skipping, which is amenable to certain mutations only, and (ii) constitutive expression of a cDNA coding for a functional, reduced-size dystrophin protein (“microdystrophin” also known as “minidystrophin”).
Both strategies, use of small antisense sequences or use of microdystrophin, address the major hurdle for the use of AAV vectors in DMD gene therapy, which is their packaging capacity. AAV vectors can accommodate about 4.7 kb while the size of the wild type dystrophin cDNA is about 14 kb. To overcome this issue, a number of studies have developed partially deleted but highly functional dystrophin genes, which can be successfully packaged inside AAV vectors and were shown to improve, though not completely normalize, the dystrophic phenotype in animal models.
The mdx mouse model is commonly used to test new constructs encoding microdystrophins. However, this model has drawbacks because the mdx mouse displays a less severe form of the disease, without immune reactions. The other animal model is the GRMD dog, which is considered more reliable to predict the therapeutic potential of a gene therapy product in humans (Kornegay et al., Mamm Genome, 2012).
Among all the proposed microdystrophin sequences, Foster H. et al. (Mol Ther, 2008. 16(11): p. 1825-32) compared in mice two different configurations of microdystrophin genes, ΔAB/R3-R18/ΔCT and ΔR4-R23/ΔCT, under the control of a muscle-specific promoter (Spc5-12) in a recombinant AAV vector (rAAV2/8). It was reported that codon human optimization of microdystrophin improved gene transfer and muscle functions in the mdx mouse model. Intravenous injection of 3·1011 vg total of rAAV/8 allowed efficient cardiac gene transfer and marked dystrophin expression in the skeletal muscle and within the diaphragm.
In relation with CXMDj dogs, Ohshima S. et al. (Mol Ther, 2009. 17(1): p. 73-80) reported the administration into dogs of a rAAV8 encoding a M3 microdystrophin under the control of the CMV promoter by limb perfusion, i.e. intravenous injection under pressure.
Zhang Y. et al. (Hum Mol Genet, 2013. 22(18): p. 3720-29) studied the systemic (5·1012 vg total) dual AAV9 gene therapy in DMD mice. By homologous recombination, the dual AAV vectors injected via the tail vain reconstituted a nNOS binding microdystrophin containing dystrophin repeats R16 and R17.
Similarly, Odom G. et al. (Mol Ther, 2011. 19(1): p. 36-45) demonstrated reconstitution of an expression cassette encoding a ΔH2-R19 minidystrophin in mice following intravascular co-delivery of two rAAV6 vectors (2·1012 vg total) sharing a central homologous recombinogenic region.
Wang B. et al. (J Orthop Res. 2009; 27(4): p 421-6) disclosed the intraperitoneal (i.p.) injection of 3·1011 vg total rAAV1 vectors in neonatal mice (dKO and mdx). These AAV vectors encode the microdystrophin Δ3990 placed under the control of the MCK or CMV promoter.
Koppanati et al. (Gene therapy. 2010; 17(11): p 1355-62) reported in utero gene transfer in the mdx mouse via the intraperitoneal (i.p.) injection of 6.4·1011 vg total rAAV8 vector encoding a canine microdystrophin placed under the control of the CMV promoter.
Schinkel et al. (Human Gene therapy. 2012; 23(6): p 566-75) reported cardiac gene therapy in the mdx mouse via the intravenous (IV) injection of 1012 vg total rAAV9 vector encoding a microdystrophin placed under the control of the CMV promoter or the cardiac-specific MLC0.26 promoter.
Gregorevic et al. (Mol. Therapy 2008; 16(4): p 657-64) reported muscular gene therapy in the mdx mouse via the intravenous (IV) injection of 1013 vg total rAAV6 vector encoding the ΔR4-R23/ΔCT microdystrophin placed under the control of the CMV promoter.
Shin et al. (Gene Therapy 2011; 18(9): p 910-19) reported cardiac gene therapy in the mdx mouse via the intravenous (IV) injection of 3·1012 vg total rAAV9 vector encoding a microdystrophin (hΔCS2) placed under the control of the CMV promoter.
Shin et al. (J. of Gene Medicine 2008; 10(4): p 449) compared the delivery efficiency in mice of rAAV8 encoding the ΔCS2 microdystrophin placed under the control of the CMV promoter, by subcutaneous injection or intravenous injection.
Colgan et al. (Mol. Therapy 2014; 22(S1): p S197) reported the microdystrophin and follistatin combinatorial gene delivery by intravenous injection of rAAV6 vectors in dKO mice.
In the context of DMD, a valuable therapeutic solution would be a gene therapy product having the following characteristics:                A product which can be systemically administered, at a reasonable dose (i.e. a proper gene transfer in the target tissues) and possibly by a unique injection;        A product which is has acceptable toxicity at that dose, and especially does not induce an adverse immune response against the dystrophin protein;        A product having a satisfying tropism, i.e. a wide spread gene transfer on large territories of skeletal muscles, but also diaphragm and myocardium;        A product able to ameliorate the dystrophic disease in humans.        
In practice, previous reports have revealed that it is a very challenging task and several attempts have failed:
Studies using AAV2/6 vectors encoding a human-specific, but not codon-optimized, microdystrophin (ΔR4-R23/ΔCT) under a CMV promoter resulted in the limited expression and eventual destruction of injected CXMDj dog muscle fibers via the immune system at 6 weeks after discontinuation of immunosuppression, 22 weeks after initial intramuscular injection (Wang, Z. et al., Mol Ther, 2007. 15: p. 1160-66).
Clinical trials based on the intramuscular injection of AAV2/5 vectors encoding a human-specific, but not codon-optimized, microdystrophin (ΔR3-R21/ΔCT) under a CMV promoter resulted in very limited transgene expression and in an inappropriate immune response (Mendell, J R et al., N Engl J Med, 2010. 363(15): p. 1429-37; Bowles, D E. et al., Mol Ther, 2012. 20(2): p. 443-55).
Therefore, there is a need in the art for an efficient treatment of dystrophic pathologies in humans, including systemic benefits in terms of survival, overall clinical score, cardiac and/or respiratory function.